PROJECT SUMMARY (30 LINES) Ischemic stroke is the fifth leading cause of death in the United States, and one of the leading causes of long- term disability in adults. One of the hallmarks of ischemic stroke is cell swelling, which occurs primarily in astrocytes. In order to regulate their volume, cells engage volume regulated anion channels (VRAC), which are chloride channels that are permeable to a number of organic osmolytes, including glutamate. Overactivation of glutamate receptors causes an excitotoxic cascade that culminates in calcium-mediated neuronal damage. This theory of swelling-activated glutamate release in the ischemic brain has been around for several decades, as inhibitors of VRAC (tamoxifen and DCPIB) reduce stroke volumes and intraischemic glutamate release. However, the lack of both knowledge on the molecular identity of VRAC and specificity of VRAC inhibitors prevented further research in this field. The recent discovery that VRAC is composed of proteins from the LRRC8 family, with subunits LRRC8A-E, has opened the doors for new studies using molecular biology tools to specifically target VRAC and the essential LRRC8A subunit. In the following work, we plan to establish LRRC8- containing VRAC as a primary contributor to intraischemic glutamate release and provide a mechanism for the neuroprotective effects of nonspecific VRAC inhibitors. In preliminary studies, we have shown in vitro that heterogenous populations of VRAC are preferentially permeable to glutamate, and that VRAC channels are likely active in the ischemic penumbra and possibly even the ischemic core. For the proposed in vivo experiments, we will use transgenic floxed LRRC8A mice to develop an astrocyte-specific LRRC8A knockout (KO) mouse model. First, we will determine if the neuroprotective properties of VRAC inhibitors are due to their inhibition of VRAC activity by measuring stroke volumes and behavior outcomes in astrocytic LRRC8A KO mice following permanent distal occlusion of the middle cerebral artery, a mouse model of stroke. We will also test whether LRRC8-containing VRAC is a major pathway for intraischemic glutamate release, using microdialysis techniques to measure cortical glutamate from astrocytic LRRC8A KO animals. This work will help us to understand more about the complex mechanisms of damage in the ischemic brain, and someday allow for the development of better therapeutics for ischemic stroke. Through the proposed work and training over the next two years, I will hone my knowledge and skills in animal models of disease and prepare myself to become a successful, independent scientist in the field of pharmaceutical research. With the help of the Ruth L. Kirschstein National Research Service Award (NRSA) Individual Predoctoral Fellowship, I will be able to design and perform research establishing a new target for ischemic stroke treatment, and will be ideally suited for future work with clinical trial development for patients suffering from neurodegenerative diseases.